The invention generally relates to mass spectrometers and specifically to tandem mass spectrometers. More specifically, the invention provides an effective coupling of a first time-of-flight mass spectrometer to a second mass spectrometer of any one of various types, including a time-of-flight mass spectrometer with orthogonal acceleration, through use of a collision cell with collisional damping.
Mass spectrometer (MS) instruments analyze compounds and their mixtures by measuring the mass to charge ratio (M/Z) of ionized molecules generated at a source. Time-of-flight (TOF) mass spectrometers accelerate a pulsed ion beam across a nearly constant potential and measure the flight time of ions from their origination at the source to a detector. Since the kinetic energy per charge of an ion is nearly constant, heavier ions move more slowly and arrive at the detector later in time than lighter ions. Using the flight times of ions with known M/Z values, the TOF spectrometer is calibrated and the flight time of an unknown ion is converted into an M/Z value.
Historically, TOF mass spectrometers have been primarily used with pulsed sources thereby generating a discrete burst of ions. Typical examples of mass spectrometers with pulsed sources include plasma desorption mass spectrometers and secondary ionization mass spectrometers. Recently TOF mass spectrometers have become widely accepted, particularly for analysis of labile biomolecules and other applications requiring wide mass range and high speed, sensitivity, resolution and mass accuracy. New ionization methods such as matrix assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) have greatly extended applications of TOF mass spectrometry. TOF mass spectrometers have become one of the most preferred instrumentation platforms for both of these new ionization methods.
The pulsed nature of the MALDI ion source naturally complements the pulsed operation of a time-of-flight analyzer, and thus TOF has been the mass spectrometer of choice from the earliest applications of the MALDI method. However, early MALDI implementation suffered from extreme sensitivity to laser energy. Recently, the resolution of MALDI/TOF MS instruments has been significantly improved by using a delayed ion extraction (DE) method, as described in U.S. Pat. Nos. 5,625,184; 5,627,369; and 5,760,393. In this method, a plume of ions and neutral molecules is allowed to expand after desorption by a laser shot and then the ions are accelerated after application of a delayed electric pulse. As a result, ions are no longer dragged through the dense plume by a high electric field. This technique reduces the energy spread of the ions and the amount of fragmentation. The delayed ion extraction method is much less sensitive to laser energy, and much higher resolution and mass accuracy are routinely available with MALDI-TOF mass spectrometers.
While pulsed sources are readily adapted to TOF mass spectrometers, it is more difficult to apply TOF to intrinsically continuous sources, like ESI. The problem was resolved with the introduction of an orthogonal extraction scheme, as described in Russian Patent SU1681340A1 and corresponding Published PCT application W091/03071, entitled xe2x80x9cMethod of time-of-flight analysis of continuous ion beamxe2x80x9d. In orthogonal TOF (o-TOF) MS instruments, a continuous, slow-moving ion beam is converted into ion pulses by means of an orthogonal pulsed electric field. Ion pulses are accelerated in a direction orthogonal to the ion beam path to a much higher energy and are focused onto an intermediate focusing plane, which serves as an object plane of a reflecting TOF MS. The orthogonal pulser/accelerator serves as a high repetition rate (typically 10 kHz) pulsed ion source for the o-TOF mass spectrometer. The efficiency of conversion, referred to as the xe2x80x9cpulser duty cyclexe2x80x9d, is usually in the order of 10 to 20%. The conversion losses are well compensated by the ability of TOF mass spectrometers to detect all ions in a given pulse. As a result, the orthogonal TOF scheme provides a significant improvement in sensitivity compared to traditionally used scanning instruments, such as quadrupole and magnet sector spectrometers, which transmit only one narrow M/Z component at a time and discard the rest of the ion beam. The acquisition duty cycle of scanning instruments (i.e., the portion of the ion beam used for analysis considering that only a single component is passed at a time) is inversely proportional to mass resolution and is in the order of 10xe2x88x924 to 10xe2x88x923%, compared to an acquisition duty cycle of xcx9c10% for o-TOF MS instruments. In addition to high sensitivity, the o-TOF scheme provides greater mass range, exceptional speed, medium to high resolution and high mass accuracy.
While ESI-TOF MS and DE MALDI-TOF MS provide excellent data on the molecular weight of samples, one disadvantage to these instruments is that they provide little information on molecular structure. Traditionally tandem mass spectrometers (MS-MS) have been employed to provide structural information. In MS-MS instruments, a first mass spectrometer is used to select a primary ion (or ions) of interest, for example, a molecular ion of a particular compound, and that ion is caused to fragment by increasing its internal energy, for example, by colliding the ion with neutral molecules. A second mass spectrometer then analyzes the spectrum of the fragment ions, and often the structure of the primary ion can be determined by interpreting mass spectra of fragment ions. The MS-MS technique improves recognition of a known compound with a known pattern of fragmentation and also improves specificity of detection in complex mixtures, where different components give overlapping peaks in the first MS instrument. In the majority of applications, such as drug metabolism studies and protein recognition in proteome studies, the detection level is limited by chemical noise. Frequently, the MS-MS technique improves the detection limit in such applications.
In MALDI-TOF MS, the technique known as post-source decay (PSD) can be employed in a single MS instrument to provide information on molecular structure. The primary ions are separated in space in a linear TOF mass spectrometer and are selected by a timed ion selector. Ions are excited during the ion formation process and partially fragment in a field-free region (referred to as metastable fragmentation). Fragment ions continue to fly with the about the same velocity and, hence, with energy proportional to their mass (known as the energy partitioning effect). Subsequently, the ion fragments can be time separated in an electrostatic mirror (reflector). The PSD method, although involving a single mass spectrometer, is referred as a pseudo MS-MS scheme. Fragmentation spectra are often weak and difficult to interpret. Adding a collision cell where ions may undergo collision induced dissociation (CID) improves fragmentation efficiency. Still, the performance of both PSD and CID spectra is strongly affected by energy partitioning and, in the CID case, by an additional collisional energy spread. Parent ions and fragment ions have different energies and thus can not be simultaneously focused in a reflecting TOF mass spectrometer with an electrostatic ion mirror. To resolve the problem the mirror voltage is stepped and the spectrum is composed of stitches, a practice which hurts sensitivity, acquisition speed and mass accuracy.
Nowadays, the most common form of tandem mass spectrometer is a triple quadrupole (Triple Q), where both mass spectrometers are quadrupoles and the collision cell uses a radio frequency (RF)-only quadrupole to enhance ion transport. Because of its low scanning speed the Triple Q instrument employs continuous ion sources such as ESI and atmospheric pressure chemical ionization (APCI) sources. Since scanning of the second mass spectrometer would cause additional losses, the most effective way of using a Triple Q instrument is in monitoring selected reactions. Drug metabolism studies are a good example where a known drug compound is measured in a rich biological matrix, such as blood or urine. In those studies both parent ion and daughter fragment ion masses are known and the spectrometer is tuned to detect those specific masses. For more generic applications requiring scanning, the triple quadrupole instrument is less desirable because of its low speed, sensitivity, mass accuracy and resolution.
In the development of triple quadrupole instruments, use of the collision cell was perfected, thereby enabling these instruments to achieve significant commercial success. The low energy collisions provide a well-controlled degree of fragmentation and significant structure information. The RF-only quadrupole guide provides complete radial retention of the ion fragments. Collisional cooling in the cell confines ions onto the axis of the cell and strongly reduces axial energy spread, as described in U.S. Pat. No. 5,248,875.
Recently hybrid instruments have been described having a quadrupole as the first MS instrument and where the second quadrupole mass spectrometer is replaced by an o-TOF mass spectrometer. This instrument is commonly referred to as a xe2x80x9cQ-TOFxe2x80x9d. The o-TOF back end permits the observation of all fragment ions of interest at once and the acquisition of secondary spectra at high resolution and mass accuracy. In cases where the full mass range of daughter ions is required, for example, for peptide sequencing, the Q-TOF instrument affords significant performance advantages over the triple quadrupole instrument. However, the Q-TOF instrument exhibits a 10 to 100 loss in sensitivity compared to the use of a single quadrupole operating in a selected reaction monitoring mode (i.e., monitoring a single M/Z value). For the same reason the sensitivity of the Q-TOF is lower in the mode of xe2x80x9cparent scanxe2x80x9d where, again, the second MS instrument is used to monitor a single M/Z value. Recently the Q-TOF platform has been applied in combination with a MALDI ion source as published by Standing et al in Rapid Comm. Mass Spectrom. 12, 508-518 (1998).
In another recent variation, it has been proposed to configure an MS-MS instrument by combining a linear ion trap (LIT) and a TOF spectrometer. A LIT is formed by modifying a conventional quadrupole with electrostatic xe2x80x9cplugsxe2x80x9d and is capable of trapping ions for a long period of time. The quadrupole field structure enables the application of various separation and excitation methods, previously developed in 3-D ion trap technology. While the LIT eliminates ion losses at selection and also can operate at poor vacuum conditions thereby reducing the requirements on the pumping system, it does suffer from limited resolution (R) of ion selection, with R less than 200 only being demonstrated at the present time.
Lately, a MALDI ion source has been coupled to a three-dimensional (3-D) quadrupole ion trap mass spectrometer (IT MS). The IT MS is a routine tool for tandem mass spectrometric analysis, providing moderate performance of individual mass spectrometric steps, but having an advantage of multiple step tandem-MS analysis, usually referred as MSn analysis. In such analysis a pulse of primary ions is trapped in the ion trap cell and is subjected to a timed sequence of operations. Those operations include selection and fragmentation of primary ions, with subsequent ejection of unwanted components, followed by selection and fragmentation of a single fragment ion of the next generation. After n steps of selection and fragmentation, the fragments are mass analyzed. Coupling the MALDI source to the IT MS has been problematic in conducting analyses using this technique. Ions produced in a MALDI source at vacuum are transported via an electrostatic lens and trapped in an IT MS cell, using an RF field with a slowly ramped amplitude. Such method of coupling introduces a significant decay of primary ions. The method works only in combination with so-called xe2x80x9csoftxe2x80x9d matrices. Since the trap is filled with ions of all masses, including matrix ions, space charge effects, including discrimination and mass shift, become pronounced. The cycle of ion storage and mass analysis is slow, the usual repetition rate of the laser is 2 Hz, and the sample is poorly utilized. Additionally, the method is sensitive to laser energy and depends on choosing an appropriate sweet spot on the sample deposited on the matrix.
High sensitivity, resolution and mass accuracy are important characteristics of TOF mass spectrometers. This is particularly true for a DE MALDI source operating in vacuum, where the ion beam already has a short duration and also has low divergence and energy spread. The transmission of TOF mass spectrometers is close to unity. Therefore, in the case of pulsed ion sources it is desirable to utilize a TOF mass spectrometer for each analyzer that forms a portion of the tandem mass spectrometer.
To overcome problems encountered in collision cells used in prior DE MALDI TOF mass spectrometers associated with the energy partitioning effect and the inability to focus all fragment ions simultaneously (see above description of PSD method), it has been proposed to add a second DE source after the collision cell, as described in co-pending patent application, Ser. No. 09/233,703, entitled xe2x80x9cA tandem mass spectrometer with delayed extraction and method of usexe2x80x9d, commonly assigned as with the present application.
In that patent application, the primary ion beam is separated in a linear TOF mass spectrometer and ions of a particular mass-of-interest are selected by a timed ion selector. The primary beam is time focused onto a plane of the ion selector, thereby enhancing the resolution of selection. The selected ion beam is directed into a collision cell, where ions experience one to a few high-energy collisions. Based on the fact that ions of interest have a much higher mass than the gas molecules with which they collide, the ion beam still preserves most of its original direction and time pulse properties. The energy of fragments still depends on mass, but because of the medium energy (1 to 3 keV) of the initial beam the energy spread is limited. After exiting the collision cell, ions are accelerated after an appropriate time delay by a second electric pulse as in DE MALDL The second acceleration increases ion energy substantially; however, the energy spread remains within the energy-focusing properties of the electrostatic mirror, known to handle an approximate 10% energy spread without loss of resolution.
While the scheme described in this patent application is expected to provide unique information concerning high energy CID and to generate maximum possible sensitivity for MALDI MS-MS experiments, high-energy collisions produce a wide spectrum of excitation and could generate a larger amount of small mass fragments. The necessity of synchronization of both TOF mass spectrometers adds a degree of complexity to the operation of this instrument. Also, the focusing properties of the second mass spectrometer take into account the focusing conditions of the first mass spectrometer and the timed ion selector.
Despite the activities to expand the capabilities of mass spectrometry outlined above, the need still exists for an improved tandem mass spectrometer that incorporates the high sensitivity, resolution and mass accuracy of TOF mass spectrometers and that is capable of utilizing to full advantage intrinsically pulsed ion sources, such as MALDI, with minimal loss of sensitivity. It is also desirable to combine the most sensitive TOF mass spectrometer with a low energy collision cell to control the degree of fragmentation and to increase the yield of information containing middle-mass fragments, while improving the energy and angular spread of the ion beam exiting the energy adjusting electrodes to improve performance of the second mass spectrometer and to decouple its operation from the first mass spectrometer.
The invention overcomes the disadvantages and limitations of the prior art by providing a high performance mass spectrometer and MS method employing time-of-flight separation of primary ions, which matches the pulsed nature of practically important pulsed ion sources, in particular a MALDI ion source.
A feature of the present invention includes coupling a time-of-flight mass spectrometer to energy adjusting electrodes with a gas at sufficiently high pressure that produces multiple collisions between the ions and the background gas to substantially damp the kinetic energy of the ion beam. In accordance with another feature of the invention, an RF multipole is included in the collision cell to spatially confine the beam. In addition, the kinetic energy of ions injected into the cell (also referred to below as xe2x80x9cinjection energyxe2x80x9d) may be adjusted by regulating static voltages or by applying electric pulses (also referred to below as xe2x80x9cdynamic energy correctionxe2x80x9d) to control the degree of fragmentation in the cell. In the particular case of low energy injection, the primary ions remain intact, and in the case of higher energy injection, the ions fragment in the collision cell. This feature allows switching between MS and MS-MS analysis while using the second MS for data acquisition. The pulsed nature of the primary beam may be partially preserved to enhance sensitivity of tandem MS operation.
The most general preferred embodiment of a tandem mass spectrometer of the invention includes a pulsed generator of ions coupled to a time-of-flight mass spectrometer, a timed ion selector, a collision cell with a gas of sufficiently high pressure to collisionally damp the admitted ion beam and to induce fragmentation in communication with the time-of-flight mass spectrometer and the timed ion selector, and a second mass spectrometer to analyze fragment ions.
In one preferred embodiment of the invention, a tandem mass spectrometer includes a DE MALDI ion source, a linear TOF MS with a timed ion selector, energy adjusting electrodes and a differentially pumped collision cell, an RF-only multipole within the collision cell, and an orthogonal TOF MS as the second MS. The energy adjusting electrodes utilize electric pulses to adjust the injection energy at a given potential on the sample plate. The cell is filled with gas to about 10 to 100 mtorr pressure to convert a pulsed, medium-energy beam into a slow quasi-continuous beam, confined near the axis of the cell by the RF field. The resultant continuous, slow ion beam is analyzed in the o-TOF mass spectrometer pulsing at high frequency, asynchronously from the operation of the first TOF mass spectrometer.
The invention can be embodied with multiple features, which taken singularly or in combination, enhance the performance of the MS instrument and method.
In one particular feature, the MALDI source employs a high repetition rate laser operating at an increased laser energy. This provides for higher sensitivity.
In another particular feature, the resolution of the TOF primary ion selection is improved for operation at elevated laser energy by introducing a second, corrective decelerating electric pulse in the first TOF MS to enhance time-of-flight resolution around the selected ion mass of interest.
Yet, in another particular feature, the timed ion selector is a time-synchronized pulsed accelerator, accelerating ions of interest only. This permits passing through only ions of a predetermined M/Z value to enhance resolution of ion selection.
Yet, in another particular feature, an additional annular detector is used to detect the ion beam reflected by the timed ion selector in order to obtain spectra of parent ions.
Yet in another particular feature, the injection energy to induce fragmentation of selected ions is adjusted independently of parameters in the first TOF mass spectrometer by including a normally field free region between the timed ion selector and a collision cell. A voltage pulse is applied to the ions of interest as they are passing through the normally field free region to regulate the kinetic energy of the detected ions prior to entering into the collision cell.
Yet, in another particular feature, the quality of spectra derived in MS only mode of operation is improved by increasing the pressure in the collision cell between 0.1 to 1 torr. Higher gas pressure improves cooling of ions after being excited in the ion source.
Yet, in another particular feature, sensitivity is improved by filling the collision cell with a light gas such as methane. This allows injecting ions into the collision cell at higher energy and thus improving sensitivity.
Yet, in another particular feature, sensitivity is improved by introducing into the collision cell a dual cell composed of two segments, the first segment being a high-order multipole having a relatively large inscribed radius, and the second being a smaller-size radius quadrupole.
Yet, in another particular feature, the asynchronous operation of the two TOF mass spectrometers is improved by smoothing the time characteristics of the ion beam by introducing a slight retarding potential at the exit end of the collision cell.
Other types of mass spectrometers may be used as the second MS analyzer, for example 3-D ion trap, Fourier transform, quadrupole or magnet sector mass spectrometers. This embodiment can utilize the time characteristic smoothing enhancement mentioned above.
In another preferred embodiment, a short collision cell operated at a higher gas pressure provides a degree of energy damping while still preserving the pulsed nature of the beam. In one mode of operation, the second mass spectrometer, an o-TOF MS, is synchronized with the ion source and the first TOF mass spectrometer to eliminate duty cycle losses.
In another embodiment, a continuous ion source, for example an ESI or APCI source, is converted into pulsed ion packets to function as a pulsed ion generator. The beam is spatially focused to reduce the size of apertures in the collision cell.
The invention also relates to a method for tandem mass spectroscopy. The method includes generating a pulse of ions from a sample of interest in a time-of-flight mass spectrometer. Ions of interest are selected from the pulse of ions in the time-of-flight mass spectrometer. The selected ions are collided with a gas having a sufficiently high gas pressure to substantially dampen the kinetic energy of the selected ions and inducing fragmentation of the selected ions. The selected ions and fragments thereof are then analyzed with a second mass spectrometer.
In one embodiment, the invention relates to a method of high performance tandem mass spectrometry which includes generating a pulsed acceleration of an ion beam from a pulsed ion source; directing the ions into a time of flight mass spectrometer; selecting only parent ions of a predetermined M/Z value for further analysis; introducing the beam of selected ions into a collision cell with an RF-only multipole at a controlled energy and pressure, where the pressure is adjusted to provide complete damping of the kinetic energy of the ions and to achieve a desired degree of fragmentation; and analyzing the fragment ions in a second mass spectrometer. This method of tandem mass spectrometry may also include preserving the pulsed nature of the primary ion beam to enhance sensitivity of the second o-TOF mass spectrometer.
One feature of the above method includes switching between MS-only and MS-MS modes by switching xe2x80x9conxe2x80x9d and xe2x80x9coffxe2x80x9d the timed ion selector and also by controlling the kinetic energy of ions injected into the collision cell. The second mass spectrometer is used to acquire spectra at all individual steps, such as acquisition of parent spectra, monitoring the quality of ion selection and acquisition of fragment ion spectra.
Coupling a TOF mass spectrometer to a low-energy collision cell followed by tandem mass spectrometry analysis by a second analyzer introduces a number of technical challenges, such as increased gas load, ion beam focusing at the entrance of the cell, and preservation of the pulsed nature of the ion beam or smoothing of the beam in the collision cell. As a result, the present invention represents a technical advance by solving these challenges in an unusual way or by an unusual combination of elements. These multiple useful variations of individual components will be discussed more fully in the following detailed description of the invention and in the accompanying experimental section.
In particular, it is an object of the invention to minimize the effect of the primary TOF mass spectrometer and the collision cell on performance and operation of the second mass spectrometer, when the invention is used for tandem MS analysis. It is also an object of the invention to enable fine control over the fragmentation process in a tandem mass spectrometer.